Paraoxonase 2 serves a proapopotic function in mouse and human cells in response to the Pseudomonas aeruginosa quorum-sensing molecule N-(3-Oxododecanoyl)-homoserine lactone

Abstract

Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKOR MEF): nuclei fragmented; mitochondrial membrane potential (Δψmito) depolarized; Ca(2+) was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca(2+)] (Cacyto); and caspase 3/7 was activated. DKOR MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKONR MEF). RNAseq analysis, quantitative PCR, and Western blots showed that WT and DKOR MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), whereas DKONR MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKONR MEF rendered them responsive to C12: Δψmito depolarized, Cacyto increased, and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control Δψmito, Ca(2+) release from the ER, and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin and staurosporine, but activates Bax- and Bak-independent apoptosis in response to C12.

Keywords: Apoptosis; Bax; Cancer; Paraoxonase 2; Pseudomonas aeruginosa (P. aeruginosa); Quorum Sensing.

FIGURE 1.

Expression of Bak and Bax in WT but not in DKO_NR or DKO_R MEF. A, Q-PCR. B, Western blot. *, p < 0.05 versus WT. Data are typical of three different experiments.

FIGURE 2.

Staurosporine and etoposide activation of apoptosis requires Bak or Bax expression in MEF. A, DKO_NR MEF or DKO_NR MEF retrovirally re-expressing GFP, Bak, Bax, or Bak + Bax were left untreated or were incubated with staurosporine (10 μm) for 2 h, followed by assay for caspase 3/7 activity. Data are averages ± S.D. (n = 3). B, DKO_NR MEF or DKO_NR MEF retrovirally re-expressing GFP, Bak, Bax, or Bak + Bax were left untreated or incubated with etoposide (10 μm) for 24 h, then exposed to PI and processed by FACS to measure cell viability. *, p < 0.05 versus control.

FIGURE 3.

C12 induces apoptosis in WT and DKO_R, but not DKO_NR MEF. A, WT, DKO_NR, and DKO_R MEF were left untreated (control) or treated with C12 (50 μm) for 2 h followed by processing for caspase 3/7 activity. Data are averages ± S.D. (n = 3). B, WT, DKO_NR and DKO_R MEF were left untreated (control) or treated with C12 (100 μm) for 24 h, followed by adding PI and processing in the FACS. *, p < 0.05 versus control.

FIGURE 4.

RNAseq summary of gene expression in WT, DKO_NR, and DKO_R MEF. Numbers of genes expressed differently in the different groupings are shown.

FIGURE 5.

Expression of PON2 in WT and DKO_R but not in DKO_NR MEF. A, Q-PCR. *, p < 0.05 versus WT. B, Western blot. Quantitation by densitometry (average ± S.D., n = 3) is shown at the bottom of the figure. Data are typical of three different experiments in both A and B.

FIGURE 6.

Expression of hPON2 in DKO_NR MEF renders them sensitive to C12-induced caspase activation. A, Q-PCR (relative to Rps17) of PON2 expression in adenovirus (adv)-GFP DKO_NR and adv-hPON2 DKO_NR. *, p < 0.05 versus DKO_NR + adenovirus-GFP. B, caspase 3/7 activity (in relative light units) in DKO_NR-GFP and DKO_NR-hPON2 in response to C12. DKO_NR-GFP and DKO_NR-hPON2 MEF were left untreated (control) or treated with C12 (50 μm) for 2 h followed by processing for caspase 3/7 activity. *, p < 0.05 versus DKO_NR + adenovirus-GFP. Data are averages ± S.D. (n = 3).

FIGURE 7.

C12 causes depolarization of Δψ_mito in hPON2-expressing DKO_NR MEF. DKO_NR MEF were treated with either adenovirus-hCFTR (serves as control) or adenovirus-hPON2 and loaded with JC1 (10 μm). Δψ_mito was measured from the green/red fluorescence ratio in the imaging microscope during control conditions and following addition of C12 (50 μm) and the protonophore FCCP (10 μm). A, C12 caused little effect on Δψ_mito of DKO_NR MEF that expressed little PON2, and FCCP elicited rapid, maximal depolarization. B, C12 caused slow but persistent depolarization of Δψ_mito of DKO_NR MEF that had been treated with advenovirus-hPON2, and FCCP elicited little further depolarization. C, summary of effects of C12 on Δψ_mito in DKO_NR-hPON2 and DKO_NR + hPON2 MEF. Magnitude of an increase of the JC1 ratio was measured after a 20–25-min treatment with C12 and expressed as percentage of maximal (100%) depolarization with FCCP. Average ± S.D. (n = 3, average of >50 cells in each experiment) are shown. *, p < 0.05 versus DKO_NR-PON2.

FIGURE 8.

C12 causes increases of Ca_cyto in hPON2-expressing DKO_NR MEF. DKO_NR MEF were treated with either adenovirus-hCFTR (serves as control) or adenovirus-hPON2 then loaded with fura-2 (5 μm). Ca_cyto was measured during control conditions and following addition of C12 (50 μm) and Ca²⁺-ATPase blocker thapsigargin (1 μm). Data have been presented as percent change of the fura-2 ratio as a percent of maximum in 10 mm Ca²⁺ + ionomycin. A, C12 caused little effect on Ca_cyto of DKO_NR MEF that expressed little hPON2, and thapsigargin elicited a rapid increase in Ca_cyto. B, C12 caused a rapid increase of Ca_cyto in DKO_NR MEF that had been treated with advenovirus-hPON2, and thapsigargin elicited little further increase. C, summary of effects of C12 on Ca_cyto in DKO_NR-PON2 and DKO_NR-PON2 MEF. Magnitude of the increase of the fura-2 ratio was measured after a 15-min treatment with C12 or thapsigargin. Average ± S.D. (n = 3, >50 cells in each experiment) are shown. *, p < 0.05 versus DKO_NR-PON2.

FIGURE 9.

Expression of hPON2 in HEK293T cells renders them sensitive to C12-induced cell killing. A, Western blot of PON2 in HEK293T cells and HEK293T cells expressing vector alone, wild type hPON2, and lactonase mutant hPON2(H114Q). Quantitation by densitometry (average ± S.D., n = 3) is shown at the bottom of the figure. B, cell killing (expressed as percent) of HEK293T, HEK293T (vector), HEK293T-PON2, and HEK-hPON2(H114Q) cells in response to treatment with 0 (control), 12.5, 50, and 100 μm C12 for 24 h followed by processing for PI uptake. *, p < 0.05 versus control. Data are averages ± S.D. (n = 3).

FIGURE 10.

Caspase 3/7 activity (fold-change compared with vector control) in HEK293T-vector, HEK-hPON2, and HEK-hPON2(H114Q) cells in response to C12 (50 μm, 2 h). *, p < 0.05 versus control; #, p < 0.05 versus vector or versus PON2-H114Q. Data are averages ± S.D. (n = 3).

FIGURE 11.

C12 causes depolarization of Δψ_mito in HEK293T cells expressing wild type hPON2 but no depolarization in cells expressing vector alone and less depolarization in HEK293T cells expressing hPON2(H114Q). A, results from a typical experiment show that C12 caused little effect on Δψ_mito of HEK293T-vector cells, and FCCP elicited rapid, maximal depolarization. B, C12 caused a slow, persistent depolarization of Δψ_mito of HEK293T-PON2 cells, and FCCP elicited little further depolarization. C, C12 caused a slow and less pronounced (versus HEK293T-PON2) depolarization of Δψ_mito in HEK293T-PON2(H114Q) cells, and FCCP elicited a rapid further depolarization. D, summary of effects of C12 on Δψ_mito in vector-, hPON2-, and hPON2(H114Q)-HEK293T cells. Magnitude of increase of the JC1 ratio was measured after a 15–20-min treatment with C12 and expressed as the percentage of maximal (100%) depolarization with FCCP. Average ± S.D. (n = 3–5, average of >50 cells in each experiment) are shown. *, p < 0.05 versus initial; #, p < 0.05 versus PON2.

FIGURE 12.

C12 causes ER to release Ca²⁺ in HEK293T-PON2 cells stably expressing hPON2 but not in vector control or hPON2(H114Q)-expressing HEK293T cells. Ca_cyto was measured during control conditions and following addition of C12 (50 μm) and thapsigargin (1 μm) to achieve a maximal release of Ca²⁺ from the ER. Data have been presented as percent change of the fura-2 ratio as a percent of maximum. A, C12 caused little effect on Ca_cyto of HEK293T cells, and thapsigargin elicited a rapid increase in Ca_cyto. B, C12 caused a rapid increase of Ca_cyto in HEK293T-PON2 cells, and thapsigargin elicited little further increase. C, C12 had little effect on Ca_cyto of HEK293T-hPON2(H114Q) cells, and thapsigargin elicited little further effect. D, summary of effects of C12 on Ca_cyto in HEK, HEK-hPON2, and HEK-hPON2(H114Q) cells. The magnitude of the increase of the fura-2 ratio was measured after a 2-min treatment with C12 or thapsigargin and expressed as the percentage of the maximal fura-2 ratio. Average ± S.D. (n = 3, >50 cells in each experiment) are shown. *, p < 0.05 versus initial.

FIGURE 13.

Proposed mechanism for C12-triggered, PON2-mediated activation of early events in ER and mitochondria of host cells. P. aeruginosa produces and releases C12 that enters host cells. PON2 use its lactonase activity to cleave the lactone ring of C12 into the shown derivative. In the ER, the active site of PON2 is located in the ER lumen, so the C12 derivative is produced inside the ER and then activates the inositol trisphosphate receptor to release Ca²⁺ from the ER, leading to ER stress thereby stimulating apoptosis. In the mitochondria PON2 is associated with complex III and coenzyme Q on the inner mitochondrial membrane, and the C12 derivative is predicted to be formed close to and then inhibit the electron transport chain (ETC), leading to depolarization of Δψ_mito, and also cytochrome c, leading to its release across the outer mitochondrial membrane through Bax- and Bak-independent mechanisms (shown by ?) into the cytosol to trigger caspase 3 and subsequent apoptostic processes. Because the C12 derivative is highly reactive, it is possible that other derivatives of C12 will also be formed, and these may be the direct activators of events in both the ER and mitochondria.